EP4074840A1 - Pcr-verfahren und pcr-kit zur erhöhung der allelunterscheidung - Google Patents

Pcr-verfahren und pcr-kit zur erhöhung der allelunterscheidung Download PDF

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EP4074840A1
EP4074840A1 EP20897780.1A EP20897780A EP4074840A1 EP 4074840 A1 EP4074840 A1 EP 4074840A1 EP 20897780 A EP20897780 A EP 20897780A EP 4074840 A1 EP4074840 A1 EP 4074840A1
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oligonucleotide
discrimination
pcr
dna
boosting
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French (fr)
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EP4074840A4 (de
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Jae Jong Kim
Si-Kyu LIM
In Kyung Park
Ayoung KYUNG
Bo Mi Lee
Jeounghyun RYU
Sun Ho Cha
SeungWoo BAEK
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Genotech Corp
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Genotech Corp
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    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • C12Q1/6844—Nucleic acid amplification reactions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
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    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/131—Allele specific probes
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    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/137—Amplification Refractory Mutation System [ARMS]

Definitions

  • the present disclosure relates to a method for detection of minor alleles, such as single nucleotide polymorphism (SNP) or somatic mutation wherein the specificity and sensitivity of DNA polymerase chain reaction (PCR) is increased for the alleles. More specifically, the present disclosure relates to a PCR-based SNP genotyping/somatic mutation detection technique wherein a partial or entire duplex-forming discrimination-boosting oligonucleotide (used interchangeably with "dbOligo" in the description and drawings) is added to a PCR solution for selectively amplifying an allele and acts to strongly inhibit PCR amplification when the 3' terminal base of a primer is not complementary to a template (3'-mismatched) but has no influence on PCR amplification when the 3' terminal base of a primer is complementary to a template (3'-matched).
  • SNP single nucleotide polymorphism
  • PCR DNA polymerase chain reaction
  • SNP single nucleotide polymorphism
  • PCR polymerase chain reaction
  • Real-time PCR which is a qualitative and quantitative strategic detection technique for nucleic acids, finds applications in a variety of fields including health, agriculture, food, environment, etc. Since its development in early 1990, real-time PCR has been continuously studied to overcome technical limitations, developing into a more accurate and precise technique.
  • non-specific signals are essential for the development of real-time PCR as a diagnostic technique particularly to detect cancers, pathogens, etc. False positives which are apt to appear due to non-specific signals degrade the reliability of the test. Diagnostic technologies that have to detect a trace amount of targets, such as non-invasive liquid biopsy, require enhanced PCR efficiency and specificity so as to accurately diagnose target variations present in trace amounts. As a rule, strategies for increasing the efficiency and specificity of PCR include creation of mixtures used in PCR, design of specific probes or primers, and enzyme modification.
  • PCR mixtures have been directed toward improving PCR reactions, diminishing unnecessary primer dimers, reducing generation of non-specific PCR products due to mismatches of primers with targets, and preventing PCR efficiency drops caused by high GC rates and the formation of specific higher structure.
  • additives for PCR mixtures have been suggested, as exemplified by dimethylsulfoxide (DMSO), betaine, etc. So-called “HotStart PCR” is implemented to block unwanted nonspecific amplifications caused by primers binding on unwanted target DNA at low temperatures.
  • HotStart PCR examples include the addition of a DNA polymerase (DNAP)-specific monoclonal antibody ⁇ Biotechniques (1994) 16(6):1134-1137 ⁇ , the addition of an aptamer, which is a single-stranded oligonucleotides inhibitory of the activity of DNA polymerase ⁇ US/005693502A (1997 ); J. Mol. Biol.
  • DNAP DNA polymerase
  • aptamer which is a single-stranded oligonucleotides inhibitory of the activity of DNA polymerase ⁇ US/005693502A (1997 ); J. Mol. Biol.
  • thermostable DNA polymerase is used in PCR.
  • DNA polymerases can be divided into seven different families. Usually selected for PCR are thermostable DNA polymerases in family A, such as Taq DNA polymerase and in family B, such as Pfu DNA polymerase and a group of enzymes from archaea.
  • Taq DNA polymerase The family to which Taq DNA polymerase belong is characterized by 5' ⁇ 3' nuclease (both exonuclease and endonuclease activity, also referred to as flap endonuclease or FEN1). Advantage is taken of this activity to release specific signals through the degradation of hydrolysis probes (TaqMan probes). Lacking 3' ⁇ 5' exonuclease activity, Taq DNA polymerase is very suitable for the PCR that employs a primer having a 3'-mismatch with the template DNA.
  • AS allele-specific
  • ARMS amplification refractory mutation system
  • Probes with high specificity were developed to increase PCR efficiency and specificity.
  • DNA-binding fluorophores such as SYBR green I and SYTO9
  • target sequence-specific probes have been developed. Examples of these probes include TaqMan probe (dual labelled signaling hydrolysis probe) ⁇ P Natl Acad Sci USA 88, 7276-280 (1991 ) ⁇ , molecular beacons ⁇ Methods. 25, 463-71 (2001 ) ⁇ , scorpion probes ⁇ Nat Biotechnol. 17, 804-07 (1999 ) ⁇ , LUX (light upon extension) primers ⁇ Nucleic acids research. 30, e137 (2002 ) ⁇ , and Amplifluor primers ⁇ BioTechniques. 26, 552-58 (1999 ) ⁇ .
  • primers with high specificity may be exemplified by AS-PCR (allele specific PCR) that makes discrimination even one base difference in the 3' terminus and ARMS-PCR (amplification refractory mutation system PCR) that is designed to add an additional mutant sequence plus one mutant base to the 3' terminus ⁇ Mol Cell Probes. 18. 349-352 (2004 ); Nucleic Acids Res 17. 2503-2516 (1989 ); Nat Biotechnol. 17. 804-807 (1999 ); Cytokine 71, 278-282 (2015 ) ⁇ .
  • primers composed of two separate parts such as the DPO (dual-priming oligonucleotide) of Seegene Inc. ⁇ J. Am. Chem.
  • a mutated DNA accounting for cancer is very difficult to specifically detect from clinical samples because specimens such as biological tissues, blood, feces, saliva, and so on have a trace amount of the mutated gene among a vast amount of wild-type DNA.
  • specimens such as biological tissues, blood, feces, saliva, and so on have a trace amount of the mutated gene among a vast amount of wild-type DNA.
  • diagnostic method that guarantees the specificity allowing for detecting a mutation incorporated in a trace amount of 1 % or less into a vast amount of wild-type genes, alongside high robustness for clinical applications.
  • low false-positive for wild-type sequences and high specificity for mutated sequences should be required.
  • improved ingredients of PCR mixture, specific probes and/or primers, or enzyme modification has been developed to increase PCR efficiency and specificity for use in diagnosis necessary for high sensitivity, such as in cancer diagnosis.
  • reagents for increasing amplification efficiency such as DMSO, betaine, etc., monoclonal antibodies for inhibiting DNA polymerase, and oligonucleotides, so-called aptamers
  • inhibitory of DNA polymerase activity often increase DNA amplification efficiency and repress the amplification of non-specific PCR products or the formation of primer dimers, but have difficulty in increasing discrimination of alleles.
  • AS primers or ARMS primers which are designed to discriminate alleles through the presence or absence of 3' mismatches according to mutated sequences of alleles, are generally employed to improve discrimination of alleles.
  • AS primers designed to make a mismatch on only one base
  • high PCR efficiency is obtained, but discrimination of alleles is poor.
  • ARMS primers that are designed to make mismatches on two or more bases, discrimination of alleles is higher compared to AS primers, but the PCR efficiency is poor, with the resultant reduction of a limit of detection (LOD) .
  • LOD limit of detection
  • a mutant DNA polymerase with improved ability to discriminate 3' mismatches and matches is used to increases detection specificity for mutated sequences.
  • mutant DNA polymerases often decrease in enzymatic activity and thus become poor in detection sensitivity.
  • the present disclose aims to provide a PCR buffer composition and a PCR method using same, which allow for discrimination of alleles according to a primer's 3' mismatch or match, thus exhibiting improved discrimination between and/specificity for alleles or mutated sequences.
  • an aspect of the present disclosure provides a PCR buffer comprising a double helix forming oligonucleotide (discrimination-boosting oligonucleotide, "dbOligo”), and a PCR method using same.
  • a PCR buffer comprising a double helix forming oligonucleotide (discrimination-boosting oligonucleotide, "dbOligo"), and a PCR method using same.
  • AS-PCR or ARMS-PCR can be usually conducted.
  • the DNA polymerase may perform dNTP addition in a subsequent stage although there is a 3' mismatch. Once the polymerase breaks through the 3' mismatch, PCR can be smoothly carried out from the next round cycle of PCR because of no mismatches of template.
  • PCR should be restrained in the initial step when the 3' terminal base of the primer makes a mismatch with the template.
  • the frequent progression of PCR under 3' mismatch results from the frequent erroneous reaction attributed to the initial addition of an excess of DNA polymerase (abbreviated to "DNAP") for efficient PCR.
  • DNAP DNA polymerase
  • a primer (abbreviated to "P") is hybridized with a template (abbreviated to "T 2 ”) to form a DNA (that is, P/T 2 ) with a 3' mismatch, and as a result, the concentration of a substrate and enzyme complex, that is, [P/T 2 ⁇ DNAP] increases, provoking frequent 3' mismatched PCR reaction errors.
  • the formation of [DNAP ⁇ P/T 2 ] complex can be reduced by decreasing initial enzyme input.
  • the exponential amplification of an amplicon cannot be expected. In light of the PCR feature that a closed system must be maintained for automatic processes and contamination prevention, it is very difficult to continually added a DNA polymerase during progression of the reaction.
  • composition is designed to allow a DNA polymerase to be properly used in a PCR reaction.
  • a composition particularly containing a nucleic acid, a protein, and an organic compound, which can bind reversibly to a DNA polymerase is added to a PCR solution, a PCR kit, or a PCR master mix. More particularly, a substance that does not inhibit the activity of DNA polymerase during PCR may be contained. Proteins that can combine with DNA polymerase may be proteins structurally similar to DNA (DNA mimic proteins) ( Biochemistry 53, 2865-2874 (2014 )), and nucleic acids that combine with DNA polymerases may be duplex-forming oligonucleotides and more particularly, double-stranded DNA to which a DNA polymerase readily bind.
  • the present disclosure pertains to a PCR kit for detection of a mutant, the kit comprising:
  • the PCR kit for detection of a mutant may further comprise (d) at least one template including a target DNA sequence having a potential mutation locus.
  • the first base at the 3' terminus of the forward primer corresponds to the potential mutation locus of the target DNA sequence.
  • the forward primer is an allele-specific (AS) primer or an amplification refractory mutation system (ARMS) primer.
  • AS allele-specific
  • ARMS amplification refractory mutation system
  • the discrimination-boosting oligonucleotide may be at least one selected from among a DNA duplex, an RNA/DNA hybrid duplex, a double-stranded oligonucleotide, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire DNA duplex, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire DNA/RNA hybrid duplex, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire double-stranded oligonucleotide, and an oligonucleotide capable of forming a partial or perfect hairpin duplex.
  • the discrimination-boosting oligonucleotide almost little interferes with PCR reactions and enhances discrimination of mutations such as SNP or somatic mutations.
  • the partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire duplex may be a self-complementary single strand(s) or two more oligonucleotide single strands including two or more partially or entirely complementary sequences.
  • the discrimination-boosting oligonucleotide has any sequence.
  • the mutation in the target DNA sequence is a single nucleotide polymorphism.
  • the DNA polymerase is a thermostable DNA polymerase.
  • the DNA polymerase is a wild-type or a mutant DNA polymerase.
  • the discrimination-boosting oligonucleotide has a length of 10 to 100 bases, 10 to 90 bases, 10 to 80 bases, 10 to 70 bases, or 10 to 60 bases, particularly 15 to 50 bases, 15 to 40 bases, or 15 to 30 bases (both inclusive).
  • the oligonucleotide is less than 10 bases or exceeds 100 bases in length, only an insignificant effect of enhancing discrimination of alleles is obtained.
  • the discrimination-boosting oligonucleotide has a Tm identical or greater than an annealing temperature set forth for PCR amplification.
  • the Tm of the discrimination-boosting oligonucleotide is lower than the annealing temperature, the discrimination of alleles is little enhanced.
  • the discrimination-boosting oligonucleotide has a Tm of 50-85°C. Given a range of 50-85°C, the Tm of the oligonucleotide can bring about a great improvement in discriminating alleles.
  • the PCR kit or method employs a probe, capable of fluorescence resonance energy transfer, for the template or an amplicon-binding substance, such as SYBR Green I or is adapted to identifying an amplicon by general electrophoresis.
  • the kit for detection of a mutant further comprises a probe, capable of fluorescence resonance energy transfer, for the template, for example, a fluorescence resonance energy transfer probe modified with a reporter and a quencher.
  • the kit for detection of a mutant further comprises an amplicon-binding substance such as SYBR Green I.
  • the present disclosure pertains to a method for detection of a genetic mutation, the method comprising the steps of:
  • the method for detection of a genetic mutation further comprises a step of (d) determining from the amplification curve acquired in step (c) whether the target DNA sequence includes a mutation.
  • the method further comprises providing a probe for the template in step (a) or (b).
  • the method further comprises providing an amplicon-binding substance such as SYBR Green I in step (a) or (b).
  • the first base at the 3' terminus of the forward primer corresponds to the potential mutation locus of the target DNA sequence.
  • the forward primer is an allele-specific (AS) primer or an amplification refractory mutation system (ARMS) primer.
  • AS allele-specific
  • ARMS amplification refractory mutation system
  • the discrimination-boosting oligonucleotide may be at least one selected from among a DNA duplex, an RNA/DNA hybrid duplex, a double-stranded oligonucleotide, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire DNA duplex, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire DNA/RNA hybrid duplex, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire double-stranded oligonucleotide, and an oligonucleotide capable of forming a partial or perfect hairpin duplex.
  • the discrimination-boosting oligonucleotide includes arbitrary sequence.
  • the mutation in the target DNA sequence is a single nucleotide polymorphism.
  • the DNA polymerase is a thermostable DNA polymerase.
  • the DNA polymerase is a wild-type or a mutant DNA polymerase.
  • DNA polymerase has been modified through amino acid substitution, deletion and/or insertion.
  • the discrimination of alleles can be further enhanced by employing the discrimination-boosting oligonucleotide of the present disclosure.
  • the discrimination-boosting oligonucleotide has a length of 10 to 100 bases, 10 to 90 bases, 10 to 80 bases, 10 to 70 bases, or 10 to 60 bases, particularly 15 to 50 bases, 15 to 40 bases, or 15 to 30 bases.
  • the discrimination-boosting oligonucleotide has a Tm identical or greater than an annealing temperature set forth for PCR amplification.
  • the discrimination-boosting oligonucleotide has a Tm of 50-85°C.
  • the PCR kit or the method employs a probe, capable of fluorescence resonance energy transfer, for the template or an amplicon-binding substance, such as SYBR Green I or is adapted to identifying an amplicon by general electrophoresis.
  • the discrimination-boosting oligonucleotide acts to interfere with PCR amplification when the primer's 3' terminal base mismatches the template and to enhance PCR amplification when the primer's 3' terminal base matches the template, thereby remarkably enhancing the specificity and sensitivity, compared to the absence of the discrimination-boosting oligonucleotide.
  • the PCR kit or method employing a discrimination-boosting oligonucleotide according to the present disclosure remarkably enhances specificity and sensitivity in general PCR as well as real-time PCR.
  • the PCR kit or method employing a discrimination-boosting oligonucleotide according to the present disclosure can recruit a substance capable of detecting amplicons in addition to a hydrolysis probe generating a fluorescent signal, thus making it possible to easily identify the position and amplification of complementary or non-complementary mutation loci.
  • the PCR kit or method of the present disclosure is advantageous for detecting alleles from a sample containing a mixture of trace amounts of various species.
  • the PCR kit or method of the present disclosure can easily detect a mutant gene even if it is present at a trace amount in a sample.
  • the embodiments of the present disclosure utilize molecular biological methods commonly known in the art to which the present disclosure belongs.
  • base refers to a nucleobase and is intended to encompass canonical purine and pyrimidine bases including adenine, guanine, cytosine, uracil, and thymine, whether natural or synthetic, and their analogs and derivatives, but with no limitations thereto.
  • nucleotide refers to a basic building block of nucleic acids, which is composed of a sugar, a base, and a phosphate group.
  • the sugar which is ribose or deoxyribose, has a base bonded to C-1' thereof and a phosphate group bonded to C-5' thereof.
  • nucleotide encompasses nucleotide analogs.
  • the sugar may or may not be substituted by other structural analogs. Examples of nucleic acid analogs composed of such compounds include phosphorothioate DNA, PNA (peptide nucleic acid), phosphoramidate DNA, morpholino, and LNA (locked nucleic acid), but are not limited thereto.
  • nucleic acid As used herein, the terms "nucleic acid”, “polynucleotide”, “oligonucleotide”, “oligomer” or other terms equivalent thereto are used to cover polymers of various monomers including polymers of monomers corresponding to nucleobase, i.e., polymers of monomers such as deoxyribonucleic acids, ribonucleic acids, phosphorothioate DNA, LNA (locked nucleic acid), PNA (peptide nucleic acid), etc., and polymers structurally similar thereto (e.g., morpholinos).
  • polymers of monomers corresponding to nucleobase i.e., polymers of monomers such as deoxyribonucleic acids, ribonucleic acids, phosphorothioate DNA, LNA (locked nucleic acid), PNA (peptide nucleic acid), etc.
  • polymers structurally similar thereto e.g., morpholinos
  • oligonucleotide means a short polynucleotide. Usually, an oligonucleotide is about 250 nucleotides or less, about 200 nucleotides or less, or 100 nucleotides or less in length.
  • the "oligonucleotide” may include modified oligonucleotides.
  • modification indicates any modification on nucleobases (e.g., purine analogs, pyrimidine analogs, inverted base, methylated analogs, fluoro analogs, etc.), linking regions (linkers) of nucleosides (e.g., amino (NH 2 ) linker, carboxyl linker, thiol (SH) linker, etc.), the phosphate group, 5'-terminal, 3'-terminal, or internal bases of oligonucleotides, and combinations thereof.
  • nucleobases e.g., purine analogs, pyrimidine analogs, inverted base, methylated analogs, fluoro analogs, etc.
  • linking regions (linkers) of nucleosides e.g., amino (NH 2 ) linker, carboxyl linker, thiol (SH) linker, etc.
  • the phosphate group 5'-terminal
  • template means "template nucleic acid” which is used to set the genetic sequence of new strands during replication in PCR. All of DNA strands which are naturally present, naturally occur, and are artificially synthesized, fall within the scope of the template.
  • target refers to a nucleic acid to be analyzed.
  • a PCR buffer may contain non-essential components such as DMSO, betaine, an aptamer, or an antibody in addition to essential components such as Mg ++ , and dNTP.
  • a PCR buffer may contain a discrimination-boosting oligonucleotide selected from among a DNA duplex, an RNA/DNA hybrid duplex, a double-stranded oligonucleotide, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire DNA duplex, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire DNA/RNA hybrid duplex, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire double-stranded oligonucleotide, and an oligonucleotide capable of forming a partial or perfect hairpin duplex, so as to further increase discrimination of alleles ( ⁇ Ct or ⁇ Cp, ⁇ Cq).
  • a discrimination-boosting oligonucleotide selected from among a DNA duplex, an RNA/DNA hybrid duplex, a double
  • the discrimination-boosting oligonucleotide used for PCR in the present disclosure may include oligonucleotides which are rendered to form a duplex by, for example, chemically coupling two or more single strands having partially or entirely complementary sequences through psoralen or a structurally similar compound thereto, or a chemical linker (e.g., disulfide linker, bismalemide linker, etc.), or by linking termini of the oligonucleotides or extending the oligonucleotide to form a complementary sequence, such as a hairpin loop structure, within single strands.
  • a chemical linker e.g., disulfide linker, bismalemide linker, etc.
  • a duplex is more easily formed with a single strand, which is not dispersed in a buffer, has a partially or entirely self-complementary sequence, compared to two separate strands which have partially or entirely complementary sequences to each other. Therefore, it is advantageous for accomplishing the goal of the present disclosure to form a duplex by using a chemical method for preventing physical separation of two complementary sequences or a method for making a complementary sequence within a single strand.
  • the discrimination-boosting oligonucleotide of the present disclosure for a DNA polymerase may vary in binding affinity for DNA polymerase, depending on various factors including the base sequence thereof, kinds of specific nucleic acids, or oligonucleotide length. In the present disclosure, however, the binding affinity is not limited by specific base sequences, specific kinds of nucleic acids, or a specific range of oligonucleotide length.
  • the complementary sequence suitable for forming a duplex is preferably 10 bases in length and more preferably 15 to 50 bases in length, with no specific limitations imparted to the complementary sequence and kinds of nucleic acids.
  • a PCR solution may contain discrimination-boosting oligonucleotide in an amount of 0.01-1,000 pmol, 0.1-500 pmol, 0.1-400 pmol, 0.1-300 pmol, 0.1-200 pmol, 0.1-100 pmol, or 1-80 pmol per 20 ⁇ L of the PCR solution.
  • the amount of the discrimination-boosting oligonucleotide may vary depending on the sequence of a target gene to be detected, a sample, and PCR conditions, and is not limited to specific concentration.
  • the present disclosure is applicable to PCR using a DNA polymerase, especially polymerases of family A (E. coli Pol I lineage).
  • This polymerase may be selected from DNA polymerases derived from thermophilic bacteria, particularly thermophilic eubacteria, and more particularly Thermus spp., Thermotoga spp. Thermococcus spp., Deinococcus spp., and Bacillus spp.
  • a duplex of the discrimination-boosting oligonucleotide has a melting temperature (Tm) higher than an annealing temperature of general PCR.
  • Tm melting temperature
  • the discrimination-boosting oligonucleotide has difficulty in forming a duplex, leading to a decrease in binding affinity for DNA polymerase.
  • the discrimination-boosting oligonucleotide used in the present disclosure is particularly configured to have a base sequence such that its duplex region has a Tm higher than an annealing temperature of general PCR.
  • FIG. 1 elucidates the mechanism of PCR in terms of kinetic parameters when a primer's 3' terminus makes a match (left panel, "3'-matched") or a mismatch (right panel, "3'-mismatched”) with a template.
  • the 3'-matched and the 3'-mismatched are each subjected to two conditions: (A) PCR in the absence of a discrimination-boosting oligonucleotide (dbOligo); and (B) PCR in the presence of dbOligo.
  • dbOligo discrimination-boosting oligonucleotide
  • DNAP stands for DNA polymerase
  • dbOligo for an oligonucleotide that has any sequence and is added in a double-stranded form or forms a duplex in a reaction solution
  • K 1 and “K -1” for kinetic parameters in a forward and a reverse direction, respectively, under the condition of making a match between a primer's 3' terminus and a template upon PCR in the absence of dbOligo
  • K 1d and “K -1d” for kinetic parameters in a forward and a reverse diction, respectively, under the condition of making a match between a primer's 3' terminus and a template upon PCR in the presence of dbOligo
  • K 2 and “K -2” for kinetic parameters in a forward and a reverse direction, respectively, under the condition of making a mismatch between a primer's 3' terminus and a template upon PCR in the absence of dbOligo
  • K 2d and “K -2” for kinetic
  • DNAP DNA polymerase
  • K cat is greatly different according to the 3' match (DNAP ⁇ P/T 1 ) or 3' mismatch (DNAP ⁇ P/T 2 ).
  • PCR very quickly starts when a primer's 3 terminus matches a template, compared to when a primer's 3' terminus mismatches a template ⁇ Clin Chem. 64(5) :801-809 (2018 ) ⁇ .
  • K cat /Km is approximately 100 to 1000-fold higher for 3'-match than 3'-mismatch. This is because K cat is substantially lowered (approximately 10- to 600-fold) and Km is slightly increased (up to 3-fold) for 3'-mismatch.
  • the DNAP ⁇ P/T 1 complex when a primer's 3' terminus matches a template, the DNAP ⁇ P/T 1 complex, once formed, continually perform repeated cycles of polymerization with dNTPs without detachment of the enzyme on the basis of high K cat1 until completion of the polymerization whereas the DNAP ⁇ P/T 2 complex formed for a mismatch between a primer's 3' terminus and a template frequently undergoes dissociation and association of the DNA polymerase due to low K cat2 (K cat1 >>> K cat2 ).
  • the present inventors predicted that when a primer's 3' terminus mismatches a template, K cat2 and K cat2d , although lower than K cat1 or K cat1d , might be similar to each other irrespective of the presence or absence of a dbOligo (K cat2 ⁇ K cat2d ⁇ K cat1 ⁇ K cat1d ). Contrary to the prediction, the presence of an dbOligo remarkably decreased the polymerization for 3' mismatch (3'-mismatched in FIG. 1(B)).
  • PCR using a discrimination boosting oligonucleotide (dbOligo) to increase discrimination of alleles or mutated genes was designated "STexS (SNP Typing with excellent specificity)" PCR.
  • Wild-type target template DNA and mutant target template DNA, forward primers, reverse primers, and hydrolysis probes for signal detection used in PCR are summarized in Table 1, below.
  • the forward primers are designed to match the mutated genes and mismatch 1 base pair of the wild-type genes at the 3' terminus of each of the primers.
  • Enzymes for each PCR were 2 units (0.05-0.08 ⁇ M) of Taq DNA polymerase (GenoTech).
  • PCR buffer (10 mM Tris, pH 9.0, 1.5 mM MgCl 2 , 60 mM KCl, 10 mM (NH 4 ) 2 SO 4 ) amounted to a total of 20 ⁇ l.
  • PCR was conducted using ABI 7500 Real-Time PCR System, starting at 95°C for 5 min followed by 45 cycles of 95°C for 30 seconds, 55°C for 40 seconds. Results are expressed as mean values of measurements for all trials that were tested in triplicate.
  • dbOligo including single-stranded DNA (SD), complementary double-stranded DNA (DD), and single-stranded DNA with an internal complementary sequence (hairpin DNA; HD) were added in an amount of 1 to 80 pmol according to test (Tables 2, 3, and 4).
  • Template DNAs were prepared by artificially synthesizing the sequences of SEQ ID NOS: 44, 45, 46, 47, 48, and 49, inserting the sequences into pTOP Blunt V2 (Enzynomics, Korea), transforming the plasmids into E. coli, and digesting the amplified plasmids with proper restriction enzymes. Before use, the purified plasmid DNAs were quantitated.
  • ⁇ Ct2 Ct of mutated gene in the presence of dbOligo - Ct of wild-type gene in the presence of dbOligo
  • ⁇ Ct ⁇ Ct2- ⁇ Ct1
  • Discrimination ability was tested according to amounts of the discrimination-boosting oligonucleotide (dbOligo) (Table 2 (Test Nos. 3 and 6)). Compared to DD-type dbOligo, HD-type dbOligo allowed a low deviation over repeated tests and thus was identified to be more consistent in PCR discrimination. For both cases, the discrimination ability was improved in a dose-dependent manner over the range of 10-80 pmol in a PCR solution. ⁇ Ct2 was measured to be up to 15.07 and maximum ⁇ Ct was 13.5 (Table 2; Test No. 3 (SEQ ID NOs: 25/26) and Test No. 6 (SEQ ID NO: 13)) ( FIG. 2 ).
  • ARMS PCR was also designed to increase discrimination for a primer with 3'-mismatch. Discrimination was examined for PCR using ARMS primers in the presence of a double-stranded oligomer (Table 4, Test No. 11). All the three types of ARMS primers (SEQ ID NOS: 2, 3, and 4) exhibited higher ⁇ Ct1 (6.50 - 7.67), compared to AS primer (SEQ ID NO: 1), but ⁇ Ct2 was measured to be 10.06 - 10.57 in the presence of dbOligo (SEQ ID NO: 13), implying that the double-stranded oligonucleotide, when used in the PCR, additionally improves the discrimination ( ⁇ Ct of 2.39 to 3.24). Taken together, the results indicate that the double-strand oligonucleotides can improve discrimination for 3'-mismatch in ARMS PCR as well as in AS PCR.
  • ⁇ Ct1 was measured to be, 4.48 for T790M; 10.45 for L858R; and 11.43 for V600E and ⁇ Ct was measured to be 2.28 for T790M; 2.31 for L858R; and 4.68 for V600E when the double-stranded oligonucleotide was added.
  • Modified polymerases may be used to increase the discrimination of PCR for 3'-mismatch.
  • a test for discrimination ability for 3'-mismatch was conducted using mutant (R536K) Taq DNA polymerase known to increase discrimination for 3'-mismatch (Table 4, Test No. 13; and FIG. 3 ).
  • primer 5 gcatgtcaag atcacagatt ttgggcg f.
  • primer 6 ggacccactc catcgagatt tct f.
  • primer 7 agtgtggaca acccccacgt gtgc r.
  • primer 8 ctggctgacc taaagccacc tc r.
  • primer 9 cacctcagat atatttcttc atgaagac probe 10 cggtggaggt gaggcagatg probe 11 taccatgcag aaggaggc probe 12 tagaccaaaa tcacctattt ttactg probe 13 gggacagtcg gaggactcgt aaaaacgag tcctccgact gtccc dbOligo 14 gggacagtcg gaggactcgt ctgg dbOligo 15 ccagacgagt cctcgactg tccc dbOligo 16 gggacagtcg gaggactcgt ctggaaaac cagacgagtc ctccgactgt ccc dbOligo 17 gggacagtcg gaggactcgt dbOligo 18 acacct
  • the PCR kit or method employing a discrimination-boosting oligonucleotide according to the present disclosure remarkably enhances specificity and sensitivity in general PCR as well as real-time PCR, thus making it possible to easily identify the position and amplification of complementary or non-complementary mutation loci. Therefore, the PCR kit or method of the present disclosure is advantageous for detecting alleles from a sample containing a mixture of trace amounts of various species and can easily detect a mutant gene even if it is present at a trace amount in a sample, finding a wide spectrum of applications in the gene examination of agricultural, aquatic, and livestock products and in the diagnostic medical field.

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